Remedy for hypertension

ABSTRACT

A therapeutic agent for hypertension, which comprises as an active ingredient a compound capable of inhibiting Na + /Ca 2+  exchanger 1.

TECHNICAL FIELD

The present invention relates to novel therapeutic agents forhypertension.

BACKGROUND ART

Intracellular free Ca²⁺ is an important ion for controlling thecontraction of cardiac muscle and various smooth muscles, the release ofneurotransmitters, and the expression of genes. The concentration ofsuch Ca²⁺ ion is regulated by Ca²⁺ pumps, Ca²⁺ channels and/or Na⁺/Ca²⁺exchangers (NCX) present in the plasma membrane and the sarcoplasmicreticulum membrane. Among them, Na⁺/Ca²⁺ exchangers play a particularlyimportant role in the contraction and relaxation of cardiac muscle andvascular smooth muscle (Ann. Rev. Physiol., vol. 52, p. 467 (1990)). Atpresent, three NCX genes have been isolated and identified from mammals.Moreover, it is known that NCX1 protein is expressed at high levels inthe brain, heart and kidney, NCX2 protein is expressed primarily in thebrain and also expressed, but slightly, in visceral smooth muscle, andNCX3 protein is expressed primarily in the brain and also expressed, butslightly, in skeletal muscle (Jpn. J. Circ. Res, vol. 24, no. 3, p. 101(2001); Am. J. Physiol., 272, C1250-C1261 (1997)).

As for NCX inhibitors, isothiourea derivatives such as2-[2-[4-[nitrobenzyloxy]phenyl] ethyl]isothio-ureamethanesulfonate(K-BR7943) and phenoxyaniline derivatives such as2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline (SEA0400)have been reported, and K-BR7943 has been confirmed to have efficacy inacute mycardial infarction models as well as brain and kidneyischemia/reperfusion models (J. Pharmacol. Exp. Ther., vol. 296, p. 412(2001)). However, there is no report about the application of NCXinhibitors as therapeutic agents for hypertension.

DISCLOSURE OF THE INVENTION

The inventors of the present invention investigated the NCX-inhibitingactivity of K-BR7943, SEA0400 and other compounds by using Na⁺/Ca²⁺exchangers (NCX) prepared from brain, heart and kidney. As a result, ithas been found that phenoxyaniline derivatives (including SEA0400) andphenoxypyridine derivatives selectively inhibit NCX derived from theheart and kidney, as compared with NCX derived from brain tissue.

In addition, previous studies have reported that the above-mentionedcompounds had little effect on other receptors, channels andtransporters, at a concentration sufficient to inhibit NCX (J.Pharmacol. Exp. Ther., vol. 298, p. 249 (2001)).

In view of the foregoing, phenoxyaniline derivatives and the like arefound to have high selectivity for NCX1.

With the aim of elucidating the relationship between NCX1 and diseasesor their treatment, the inventors of the present invention made furtherinvestigation in various disease models (e.g., diabetic rats,salt-sensitive hypertension models) using the above NCX1-selectiveinhibitors. As a result, it has been found that inhibition of NCX1 iseffective in reducing the blood pressure in salt-sensitive hypertensionmodels. These findings led to the completion of the present invention.

Namely, the present invention is directed to a therapeutic agent forhypertension, which comprises as an active ingredient a compound capableof inhibiting Na⁺/Ca²⁺ exchanger 1.

Further, the present invention is directed to a therapeutic agent forhypertension, which comprises as an active ingredient a 2-phenoxyanilinederivative of Formula (1):

[wherein R¹ represents a hydrogen atom or a C₁-C₆ alkoxy group, R²represents a halogen atom or a nitro group, and R³ represents a hydrogenatom or a halogen atom] or a pharmaceutically acceptable salt thereof.

BEST MODE FOR CARRYING OUT THE INVENTION

In the present invention, NCX1 inhibiting compounds are not limited aslong as they inhibit kidney-derived NCX1, preferred are those whichproduce greater than 50% inhibition at a concentration of 3 μM whenassayed by the test described later (Reference Example 3). In terms ofavoiding side effects, more preferred are compounds capable ofspecifically inhibiting NCX1.

The compound capable of specifically inhibiting NCX1 means a compoundthat does not substantially inhibit other receptors, channels andtransporters at a concentration sufficient to inhibit NCX 1. Morespecifically, for example, when used at a concentration of 3 μM, such acompound preferably does not cause greater than 50% inhibition of Ca²⁺channels, Na⁺ channels, K⁺ channels, Na⁺/H+ transporters, norepinephrinetransporters, Na⁺, K⁺-ATPase, Ca²⁺-ATPase, phospholipase A₂,phospholipase C, 5-lipoxygenase, inducible nitric-oxide synthetase,constitutive nitric-oxide synthetase, Adenosine receptors, Adrenergicreceptors, Glutamate receptors, Bradykinin receptors, LTB4 receptors orPAF receptors. It should be noted that procedures for measurement usingthese individual ion channels, enzymes and receptors are disclosed in J.Pharmacol. Exp. Ther., vol. 298, p. 249 (2001) and references citedtherein.

Examples of the compound capable of specifically inhibiting NCX1 includephenoxyaniline derivatives and phenoxypyridine derivatives.

Preferred are compounds of Formula (2):

[wherein R⁴, R⁵ and R⁶, which may be identical or different, eachrepresent a hydrogen atom or a halogen atom, X represents:

R⁷ represents a hydrogen atom, a substituted or unsubstituted C₁-C₆alkyl group or a substituted or unsubstituted C₁-C₆ alkoxy group, Zrepresents a nitro group, an amino group or a NHC(O)CH₂R⁸ group, R⁸represents a hydrogen atom, a substituted or unsubstituted C₁-C₆ alkylgroup, a substituted or unsubstituted C₁-C₆ alkoxy group, a halogenatom, a hydroxy group, a C₂-C₇ acyloxy group, NR⁹R¹⁰ or

R⁹ and R¹⁰, which may be identical or different, each represent ahydrogen atom, a substituted or unsubstituted C₁-C₆ alkyl group or anN-methyl-4-piperidinyl group, R¹¹ represents a hydrogen atom, a hydroxygroup or a C₂-C₇ alkoxycarbonyl group, Y represents a methylene group,an epoxy group, a thio group or a NR¹² group, n represents an integer of1 to 4, and R¹² represents a hydrogen atom, a substituted orunsubstituted C₁-C₆ alkyl group or a substituted or unsubstituted phenylgroup] or a pharmaceutically acceptable salt thereof.

In terms of NCX1-inhibiting activity, more preferred are2-phenoxyaniline derivatives of Formula (1):

[wherein R¹ represents a hydrogen atom or a C₁-C₆ alkoxy group, R²represents a halogen atom or a nitro group, and R³ represents a hydrogenatom or a halogen atom] or a pharmaceutically acceptable salt thereof.

In Formulae (1) and (2), a C₁-C₆ alkoxy group means a linear or branchedalkoxy group containing 1 to 6 carbon atoms. Examples include a methoxygroup, an ethoxy group, a propoxy group, an isopropoxy group, a butoxygroup, an isobutoxy group, a sec-butoxy group, a tert-butoxy group, apentyloxy group, an isopentyloxy group, a neopentyloxy group, atert-pentyloxy group, a 1-methylbutoxy group, a 2-methylbutoxy group, a1,2-dimethylpropoxy group, a hexyloxy group and an isohexyloxy group.

Examples of a substituent for the substituted C₁-C₆ alkoxy group includea chloro group, a fluoro group, a nitro group, an amino group, adimethylamino group, a carboxyl group, a methoxycarbonyl group, anethoxycarbonyl group, a phenyl group, a hydroxy group, a cyano group anda carbamoyl group.

A halogen atom refers to a fluorine atom, a chlorine atom, a bromineatom or an iodine atom.

A C₁-C₆ alkyl group means a linear or branched alkyl group containing 1to 6 carbon atoms. Examples include a methyl group, an ethyl group, apropyl group, an isopropyl group, a butyl group, an isobutyl group, asec-butyl group, a tert-butyl group, a pentyl group, an isopentyl group,a neopentyl group, a tert-pentyl group, a 1-methylbutyl group, a2-methylbutyl group, a 1,2-dimethylpropyl group, a hexyl group and anisohexyl group.

Examples of a substituent for the substituted C₁-C₆ alkyl group includea chloro group, a fluoro group, a nitro group, an amino group, adimethylamino group, a carboxyl group, a methoxycarbonyl group, anethoxycarbonyl group, a phenyl group, a methoxy group, an ethoxy group,a hydroxy group, a cyano group and a carbamoyl group.

A C₂-C₇ acyloxy group means a linear or branched acyloxy groupcontaining 2 to 7 carbon atoms, whose acyl moiety may be cyclic or maycontain an aromatic group. Examples include an acetoxy group, apropionyloxy group, an isopropionyloxy group, a cyclohexyloxy group anda benzoyloxy group.

A C₂-C₇ alkoxycarbonyl group means a linear or branched alkoxycarbonylgroup containing 2 to 7 carbon atoms, whose alkoxyl moiety may be cyclicor may contain an aromatic group. Examples include a methoxycarbonylgroup, an ethoxycarbonyl group, a propoxycarbonyl group, anisopropoxycarbonyl group, a butoxycarbonyl group, an isobutoxycarbonylgroup, a sec-butoxycarbonyl group, a tert-butoxycarbonyl group, apentyloxycarbonyl group, an isopentyloxycarbonyl group, aneopentyloxycarbonyl group, a tert-pentyloxycarbonyl group, a1-methylbutoxycarbonyl group, a 2-methylbutoxycarbonyl group, a1,2-dimethylpropoxycarbonyl group, a hexyloxycarbonyl group and anisohexyloxycarbonyl group.

Examples of a substituent for the substituted phenyl group include achloro group, a fluoro group, a nitro group, an amino group, adimethylamino group, a carboxyl group, a methoxycarbonyl group, anethoxycarbonyl group, a methyl group, an ethyl group, a methoxy group,an ethoxy group, a hydroxy group, a cyano group and a carbamoyl group.

Examples of compounds having an excellent activity for hypertension arethe compound shown below (SEA0400)

the compound shown below (SEA0064).

Moreover, to avoid side effects, such compounds preferably producestronger inhibition on NCX1 than on NCX2 and NCX3. For example, thecompounds that have smaller values of

-   -   IC₅₀ (renal cortex-derived)/IC₅₀ (brain-derived), and    -   IC₅₀ (renal cortex-derived)/IC₅₀ (cardiac sarcolemma-derived)        when measured as described later, than SEA 0400 are preferred.

It should be noted that the compounds of Formulae (1) and (2) can besynthesized according to the procedures as described in WO98/43943,WO99/20598, Japanese Laid-Open Patent Hei 10-265460, Japanese Laid-OpenPatent Hei 10-218844, Japanese Laid-Open Patent Hei 11-49752 andJapanese Laid-Open Patent Hei 11-92454.

As used herein, the therapeutic agent for a hypertension is intended tomeans a therapeutic agent for salt-sensitive hypertension, renalhypertension, essential hypertension, gestational hypertension orprimary aldosteronism.

The therapeutic agent of the present invention can be prepared as apharmaceutical composition in any desired dosage form for oral orparenteral use (e.g., tablets, pills, capsules, granules, dry syrups,injectable preparations), in combination with known carriers, diluentsand so on, as appropriate.

Solid preparations can be prepared by stirring granulation,fluidized-bed granulation or milling granulation using various additivessuch as excipients, disintegrating agents, binders, lubricants andcoating bases.

If necessary, it is also possible to add other additives such asantioxidants, coating agents, coloring agents, flavoring agents,surfactants and plasticizers.

The dose of the active ingredient in the pharmaceutical preparation ofthe present invention will vary depending on age, body weight, dosageform, and so on, but the usual dose to an adult is 0.1 to 1000 mg/day,which can be administrated once or several times a day.

The present invention will now be described with reference to thefollowing Formulation Examples and Test Examples, which are not intendedto limit the scope of the invention.

FORMULATION EXAMPLE 1

SEA0400 50 mg Lactose 40 mg Corn starch 49.75 mg   Crystalline cellulose17 mg Carmellose calcium 17 mg Hydroxypropylcellulose 5.25 mg  Magnesium stearate  1 mg Total 180 mg 

SEA0400, lactose, corn starch, crystalline cellulose and carmellosecalcium were mixed uniformly, followed by addition of a 10% aqueoushydroxypropylcellulose solution. After kneading and drying, theresulting granules were passed through a 30M sieve to give uniformgranules, which were then supplemented with magnesium stearate andtabletted into tablets.

FORMULATION EXAMPLE 2

SEA0064 50 mg Lactose 40 mg Corn starch 49.75 mg   Crystalline cellulose17 mg Carmellose calcium 17 mg Hydroxypropylcellulose 5.25 mg  Magnesium stearate  1 mg Total 180 mg 

SEA0064, lactose, corn starch, crystalline cellulose and carmellosecalcium were mixed uniformly, followed by addition of a 10% aqueoushydroxypropylcellulose solution. After kneading and drying, theresulting granules were passed through a 30M sieve to give uniformgranules, which were then supplemented with magnesium stearate andtabletted into tablets.

REFERENCE EXAMPLE 1 Method of measuring for brain microsomal Na⁺/Ca²⁺exchanger

Brain microsomes (1.5 mg/ml) obtained from 8-week-old rats werepre-treated with 160 mM NaCl-containing buffer to cause Na loading intomembrane vesicles. This suspension was diluted 50-fold with 20 μM⁴⁵CaCl₂-containing buffer to induce ⁴⁵Ca uptake, followed by dilutionwith the buffer (0° C.) to stop the reaction. The membrane vesicles wereimmediately collected on a nitrocellulose filter. Subsequently, ⁴⁵Catrapped inside the membrane vesicles was determined by liquidscintillation counting. The above assay for Na⁺/Ca²⁺ exchange activityin brain microsomes was performed according to the procedures describedin J. Biol. Chem., vol. 257, p. 5111 (1982).

REFERENCE EXAMPLE 2 Method of measuring for canine cardiac sarcolemmalvesicle Na⁺/Ca²⁺ exchanger

Canine cardiac sarcolemmal vesicles (0.5 mg/ml) were prepared bycentrifugal fractionation as described in Methods enzymology, vol. 157,p. 85 (1988) and suspended in Solution A (20 mM MOPS-Tris (pH 7.4), 160mM NaCl or KCl), followed by incubation at room temperature for aboutone hour to cause Na or K loading into the vesicles. This suspension wasdiluted 50-fold with 20 μM ⁴⁵CaCl₂-containing buffer to induce ⁴⁵Cauptake, followed by dilution with the buffer (0° C.) to stop thereaction. The membrane vesicles were immediately collected on anitrocellulose filter. Subsequently, ⁴⁵Ca trapped inside the membranevesicles was determined by liquid scintillation counting. The Na⁺/Ca²⁺exchange activity was evaluated by subtracting the value obtained for Kloading from the value obtained for Na loading. The above assay forNa⁺/Ca²⁺ exchange activity in canine cardiac sarcolemmal vesicles wasperformed according to the procedures described in J. Biol. Chem., vol.257, p. 5111 (1982).

REFERENCE EXAMPLE 3 Preparation of rat renal cortex-derived BLMVs(basolateral membrane vesicles) and method of measuring for theirNa⁺/Ca²⁺ exchange activity

(Preparation of BLMVs)

BLMVs were prepared from rat renal cortex and assayed for Na⁺/Ca²⁺exchange activity according to the procedures described in Am. J.Physiol., vol. 266, p. F785 (1994).

After being excised from rats, the kidneys were placed in ice-coldsucrose buffer (0.25 mM sucrose, 0.1 mM PMSF, 10 mM Tris-HCl (pH 7.6))and decapsulated. The isolated cortex was then finely minced in thesucrose buffer and homogenized sequentially with a Dounce-typehomogenizer and a Polytron-type homogenizer, followed by centrifugationat 2500 g for 15 minutes to collect the supernatant. Centrifugation wasrepeated at 24000 g for 20 minutes to collect the white fluffy portionof the pellet. After further addition of the sucrose buffer, thecollected fraction was homogenized with a Dounce-type homogenizer,supplemented with Percoll and then centrifuged at 30000 g for 35 minutesto collect the middle layer. After addition of buffer (100 mM KCl, 100mM mannitol, 5 mM HEPES-Tris (pH 7.4)), centrifugation was carried outat 34000 g for 30 minutes to collect the loose white pellet (BLMVs). Thepellet was further suspended in the KCl-mannitol buffer and thencentrifuged at 34000 g for 30 minutes to collect the precipitate, whichwas used for activity assay.

(Method of measuring for Na⁺/Ca²⁺ exchanger (Na-dependent ⁴⁵Ca uptake))

The BLMVs thus prepared were equilibrated at 37° C. for 10 minutes inpre-equilibration buffer (100 mM NaCl, 40 mM KCl, 1 mM MgSO₄, 10 mMglucose, 5 mM HEPES-Tris (pH 7.4)) and then centrifuged at 20000 g for 5minutes to collect the precipitate, which was then resuspended in thepre-equilibration buffer. Centrifugation was repeated to collect theprecipitate, followed by resuspension in the pre-equilibration buffer.The resulting BLMV suspension was diluted 20-fold with external medium(100 mM choline chloride, 40 mM KCl, 1 mM MgSO₄, 10 mM glucose, 5 mMHEPES-Tris (pH 7.4), 25 μM valinomycin, 10 μM CaCl₂, 1 mCi/l ⁴⁵CaCl₂) tostart uptake. After reaction at 25° C. for a given period of time, 2 mlstop solution (ice-cold 150 mM KCl) was added to stop the reaction, andthe reaction mixture was immediately filtered through an ultrafiltrationmembrane (0.45 μm nitrocellulose filter) to collect BLMVs on the filter.Subsequently, the filter was washed twice with 2 ml stop solution andthen the amount of ⁴⁵Ca trapped inside the BLMVs was determined by usingliquid scintillation method. TABLE 1 Inhibitory activity againstNa⁺/Ca²⁺ exchange (Na-dependent ⁴⁵Ca uptake) (IC₅₀ value: μM) BrainCardiac sarcolemma Renal cortex K-BR7943 11.0 7.0 6.8 SEA0400 0.2 0.090.02

Likewise, SEA0064 was also confirmed to show an inhibitory activity ofabout 0.07-fold of SEA0400, by measuring for renal cortex-derivedNa⁺/Ca²⁺ exchange activity.

TEST EXAMPLE 1

<Method>

(Creation of Dahl salt-sensitive hypertension rat model)

Dahl salt-sensitive hypertension rats (7 weeks old) were used in theexperiment. They were fed a 4% NaCl high-salt chow (0.6% for normalchow), but were allowed to drink water without any restriction. At twoweeks after high-salt loading, the animals confirmed to develophypertension were divided into the following 4 groups for use in theexperiment.

(Experimental animal groups) Group I: solvent (20% fat emulsion) GroupII: SEA0400  3 mg/kg Group III: SEA0400 10 mg/kg Group IV: SEA0400 30mg/kg(Experimental schedule)

After loading the high-salt chow for two weeks as described above, anacute experiment was performed. On the day of the experiment, thesystolic blood pressure (before administration) was measured for eachanimal under non-anesthesia conditions using a noninvasivesphygmomanometer. The animals were then administered orally with thesolvent or SEAO400(3, 10, or 30 mg/kg) and monitored over time for theirsystolic blood pressure.

(Result)

The level of systolic blood pressure before administration was 140±3,148±4, 143±3 and 142±4 mmHg in Group I, II, III and IV, respectively.The % change in blood pressure at one hour after administration was−3.8±1.0% in the solvent group, −9.5±1.8% in the SEA0400 3 mg/kg group,−12.0±0.9% in the SEA0400 10 mg/kg group and −12.7±3.4% in the SEA040030 mg/kg group. When compared with the solvent group, the 10 mg/kg and30 mg/kg SEA 0400 group both showed a significant reduction in bloodpressure.

INDUSTRIAL APPLICABILITY

The present invention enables to provide therapeutic agents forhypertension that are based on a novel mechanism of action. They areuseful for treating and preventing hypertension with fewer side effects.

1. A therapeutic agent for hypertension, which comprises as an activeingredient a compound capable of inhibiting Na⁺/Ca²⁺ exchanger 1 (NCX1).2. A therapeutic agent for hypertension, which comprises as an activeingredient a compound capable of specifically inhibiting Na⁺/Ca²⁺exchanger 1 (NCX1).
 3. A therapeutic agent for hypertension, whichcomprises as an active ingredient a 2-phenoxyaniline derivative ofFormula (1):

[wherein r¹ represents a hydrogen atom or a c₁-C₆ alkoxy group, r²represents a halogen atom or a nitro group, and r³ represents a hydrogenatom or a halogen atom] or a pharmaceutically acceptable salt thereof.4. A method for treating or preventing hypertension characterized ininhibiting Na⁺/Ca²⁺ exchanger 1 (NCX1).
 5. Use of a compound capable ofinhibiting Na⁺/Ca²⁺ exchanger 1 (NCX1) for the treatment ofhypertension.